Population studies have consistently demonstrated an inverse association between high-density lipoprotein cholesterol (HDL-C) and coronary heart disease (CHD). While genetic variation causing low or deficient HDL-C has been reported with premature CHD, few data have systematically evaluated high HDL-C states in association with longevity. This is an important issue to resolve because at least 50% of HDL-C is genetically mediated. Previously, our laboratory focused on studying the molecular basis of very low HDL cholesterol (e.g., <20 mg/dL) in association with premature CHD. During that period, the molecular cause of low HDL-C was identified in 16 of 20 unrelated pedigrees with functional mutations elucidated in 3 genes: apolipoprotein A-1 (APOA1), ATP-binding cassette AI (ABCA1) and lecithin-cholesterol acyltransferase (LCAT). In contrast, considerably less information is available in regard to the molecular basis of familial hyperalphalipoproteinemia (FHA), a phenotype characterized by very high HDL-C, including levels that exceed 100 mg/dL. This is a timely area for investigation in view of recent data demonstrating a putative role for HDL in reducing inflammation and regressing atheromatous lesions. To this end, we collected and analyzed blood and DNA samples from subjects with FHA in whom a familial history of longevity (at least 3 family members living to age 90 years and beyond) exists. While it is recognized that genome wide association studies (GWAS) provide an outstanding conduit for assessing genotype-phenotype associations at the population level, the subjects to be investigated in the present proposal are derived from biologically small families (n <10). Moreover, genomic sequencing of known candidate genes followed by SNP chip analysis (e.g., 500K) failed to reveal the molecular basis of FHA (see preliminary data, below). Recently, the use of Massively Parallel Sequencing by Synthesis (SBS) has evolved as a new and suitable approach for mutation discovery because in addition to identifying rare and splice-variants, this technology permits measurement of transcript abundance and expression levels of genes of interest. Moreover, SBS will assist in discriminating between SNPs and causative mutations. Based upon our prior success in identifying functional mutations causing HDL-C deficiency and having already ruled out mutations in known candidate genes causing FHA (see preliminary data), we believe that transcriptome resequencing using SBS provides an excellent platform to study the molecular basis of FHA in pedigrees with few family members. Therefore, the central hypothesis of this R21 proposal is that exceptionally high HDL-C is a consequence of single-gene mutations. Our overall aim is to identify novel mutations associated with the most extreme cases of FHA because elucidating the genetic underpinnings of FHA will provide the foundation to investigate and advance our understanding of the complex inverse relationship between HDL regulatory proteins and atherothrombotic disease. PUBLIC HEALTH RELEVANCE: Overall, this application seeks to identify the gene(s) responsible for extremely high levels of HDL, the "good cholesterol". We have collected blood and DNA samples from several unrelated subjects who have extraordinarily high levels of HDL cholesterol (greater than 120 mg/dL) in association with a family history of longevity. It is hoped that the identification of novel genes implicated in high HDL states that are associated with longevity will facilitate the development of novel therapies aimed at reducing heart disease.